Transgenic techniques have become a powerful tool for addressing important biological problems in multicellular organisms, and this is particularly true in the plant field. Many approaches that were impossible to implement by traditional genetics can now be realized by transgenic techniques, including the introduction of homologous or heterologous genes into plants, with modified functions and altered expression patterns. The success of such techniques often depends upon the use of markers to identify the transgenic plants and promoters to control the expression of the transgenes.
Selectable markers are widely used in plant transformation. Historically such markers have often been dominant genes encoding either antibiotic or herbicide resistance (Yoder and Goldsbrough, 1994). Although such markers are highly useful, they do have some drawbacks. The antibiotics and herbicides used to select for the transformed cells generally have negative effects on proliferation and differentiation and may retard differentiation of adventitious shoots during the transformation process (Ebinuma et al., 1997). Also, some plant species are insensitive to or tolerant of these selective agents, and therefore, it is difficult to separate the transformed and untransformed cells or tissues (Ebinuma et al., 1997). Further, these genes are constitutively expressed, and there are environmental and health concerns over inserting such constitutively expressed genes in plants which are grown outside of a laboratory setting (Bryant and Leather, 1992; Gressel, 1992; Flavell et al., 1992).
One marker that is neither an antibiotic nor a herbicide is the ipt gene from the Ti-plasmid of Agrobacterium tumefaciens. This gene encodes isopentenyltransferase, which is used in cytokinin synthesis (Barry et al., 1984). Isopentenyltransferase uses 5′-AMP and isopentenyl diphosphate to catalyze the formation of isopentenyl-adenosine-5′-monophosphate, the first intermediate in cytokinin biosynthesis. Overexpression of the ipt gene leads to elevated cytokinin levels (Medford et al., 1989; McKenzie et al., 1998; Faiss et al., 1997; Redig et al., 1996; Ebinuma et al., 1997). Cytokinins are plant hormones that play an important role in plant development by mediating a range of morphological changes (Mok and Mok, 1994; Davies, 1995; Coenen and Lomax, 1997). For example, cytokinins are able to stimulate leaf expansion and delay leaf senescence (Kuraish and Okumura, 1956; Wingler et al., 1998; Gan and Amasino, 1995). In young, dark-grown seedlings, high cytokinin levels can produce a deetiolated phenotype, resembling the morphology of light-grown seedlings with short hypocotyls, open hooks and expanded cotyledons (Chaudhury et al., 1993; Miklashevichs and Walden, 1997). Cytokinins can also release lateral buds from apical dominance, and stimulate de novo bud formation (Cline, 1991; Skoog and Miller, 1957; Sachs and Thimmann, 1967). This class of hormones thus plays a critical role in the formation of adventitious shoots. As demonstrated by Skoog and Miller (1957), high cytokinin levels can induce shoot differentiation from tobacco calli, a prerequisite for the regeneration of transgenic plants. Besides supporting tumor growth, T-DNA introduction into a plant cell can also induce regeneration of physiologically abnormal shoots from transformed protoplasts or leaf discs.
Overexpression of the ipt gene (Akiyoshi et al., 1984; Barry et al., 1984), a component of the T-DNA, leads to increased cytokinin relative to auxin, which triggers shoot regeneration (Tran Thanh Van, 1981). This overproduction of shoots can result in a phenotype of a large number of shoots (hereafter “shooty phenotype”). This phenotype can be used as a marker (Ebinuma et al., 1997). Studies using the ipt gene under the control of constitutive promoters showed that ipt overexpression causes elevated cytokinin levels in transgenic plants (Smigocki and Owens, 1988; Medford et al., 1989). A chimeric ipt gene under the control of the cauliflower mosaic virus (CaMV) promoter has been introduced into cells of potato (Ooms et al., 1983), cucumber (Srmigocki and Owens, 1989), and several Nicotiana species (Smigocki and Owens, 1988) and these transgenic cells proliferated and exhibited an extreme shooty phenotype and loss of apical dominance in hormone-free medium. Studies have shown that in plants transformed with ipt to overproduce cytokinins, the cytokinins work only locally as a paracrine hormone (Faiss et al., 1997). Grafting experiments performed with wild type tobacco plants and tobacco plants in which the ipt gene was overexpressed showed that the increased cytokinin levels remained restricted to the part of the plant that overexpressed ipt (Faiss et al., 1997).
One problem with the use of constitutively expressed ipt as a marker is that the resulting transgenic plants lose apical dominance and are unable to root due to overproduction of cytokinins (Ebinuma et al., 1997). In addition, plants which constitutively overexpress ipt possess an altered leaf morphology and delayed leaf senescence. Such plants show little root growth and poor internode elongation, display delayed leaf senescence, and are very often sterile (Mok and Mok, 1994; Klee et al., 1987; Ebinuma et al., 1997).
Ebinuma et al. (1997) developed one method to use the ipt marker to overcome the problems associated with constitutive overexpression of ipt. They developed a vector in which the ipt gene was inserted into a plasmid which included the transposable element Ac. The construct included the T-DNA (portion of the Ti plasmid that is transferred to plant cells) and the 35S CaMV promoter. This construct was transformed into A. tumefaciens. Leaf segments were inoculated with the transformed bacteria and grown on nonselective media. In rare cases, the Ac-element failed to re-integrate or integrated into a sister chromatid after its excision. Abnormal shoots with an extra shooty phenotype were selected and cultivated further for six months. From these, several normal shoots grew. Some of these were a result of the transposable element Ac having excised from the genome along with the ipt gene, as determined by DNA analysis. Some of these plants retained the other necessary markers which had also been included in the plasmid. This method therefore overcomes the problems of having a constitutively expressed ipt gene present. Unfortunately, this method requires many months of cultivation and results in only a few plants that have lost the ipt gene. Ebinuma et al. (1997) report that 6 months after infection the frequency of marker free plants was 0.032%. Furthermore, the selection of “normal” shoots from abnormal regenerants was based on a variable morphological criterion. The morphological selection also does not distinguish between plants that lost the 35S-ipt gene and chimeric plants or plants with very low ipt expression level.
The use of inducible promoters is another means that has been used to overcome the problems associated with the constitutive overexpression of the ipt gene in transgenic plants. The use of a copper-inducible promoter to regulate ipt expression led to the specific expression of the ipt gene in the roots, the major organ for cytokinin biosynthesis (McKenzie et al., 1998). In addition, regulated ipt expression by the tetracycline inducible system (Gatz et al., 1992) provided data about the biological effects of cytokinins in plants and their transport through the vascular system (Faiss et al., 1997; Redig et al., 1996). Transgenic plants carrying the ipt gene under the control of heat shock (Medford et al., 1989) and light inducible promoters (Redig et al., 1996) have also been reported. All of these systems were used to study the biological effects of cytokinins and were not used for transformation.
The CKI1 gene was recently identified (Kakimoto, 1996). Overproduction of this gene in plants results in plants that exhibit typical cytokinin responses, including rapid cell division and shoot formation in tissue culture in the absence of exogenous cytokinin (Kakimoto, 1996). The CKI1 gene can be used as a selectable marker in a manner similar to ipt, i.e., the CKI1 gene can be put under the control of a promoter and overexpressed in transgenic plant cells thereby inducing shoot formation in the absence of exogenous plant hormones. Such shoots can be excised, thereby obtaining transgenic plants. Such shoots, obtained either from cells transformed with ipt or CKI1, cannot be made to grow normally while the cells overexpress these transgenes.
The Knotted gene and Knotted-like genes are a third group of genes which when overexpressed can lead to ectopic production of adventitious shoots (Chuck et al., 1996; Lincoln et al., 1994; Matsuoka et al., 1993). These can be used as selectable markers in the same manner as the ipt and CKI1 genes. In general, any plant genes that can promote shoot regeneration and development can be used as selectable markers in the same manner as ipt, CKI1 and Knotted-like.
In addition to the use of markers to identify transgenic plants, the use of promoters to control expression of the transgenes is a normal part of such experiments. In most experiments, the transgenes are transcribed from a strong promoter, such as the 35S promoter of the cauliflower mosaic virus (CaMV). However, a more flexible gene expression system is needed to extract greater benefits from transgenic technology. Good inducible transcription systems are desired because transgenic plants with inducible phenotypes are as useful as conditional mutants isolated by traditional genetics. In this regard, several induction systems have been reported and successfully used (Ainley and Key, 1990; Gatz et al., 1992; Mett et al., 1993; Weinmann et al., 1994). Among these, the tetracycline-dependent expression systems are the most advanced (for review, see Gatz, 1996).
The glucocorticoid receptor (GR) is a member of the family of animal steroid hormone receptors. GR is not only a receptor molecule but also a transcription factor which, in the presence of a glucocorticoid, activates transcription from promoters containing glucocorticoid response elements (GREs) (for reviews, see Beato, 1989; Picard, 1993). It has been thought that the GR system could be a good induction system in plants because it is simple, and glucocorticoid itself does not cause any pleiotropic effects in plants. Nevertheless, a general and efficient glucocorticoid-inducible system using GR has not previously been constructed for transgenic plants, although it has been demonstrated that a system comprising GR and GREs could work in a transient expression system with cultured plant cells (Schena et al., 1991). On the other hand, it has been reported that the (hormonal) regulatory region (or domains) of GR could regulate the function of plant transcription factors in transgenic plants (Aoyama et al., 1995; Lloyd et al., 1994). Lloyd et al. (1994) showed that trichome development in Arabidopsis could be successfully controlled by a chimeric protein comprising the glucocorticoid regulatory domains and the maize transcriptional regulator R. However, the construction of such a chimeric transcription factor whose activity is tightly regulated by the glucocorticoid receptor domain is not always easy and achievable in every case. Tight regulation appears to be critically dependent on the intramolecular structure of the chimeric protein, especially the relative position between the glucocorticoid receptor domain and the domain whose function is to be regulated.
The regulatory region of animal steroid hormone receptors, which include a hormone binding domain (HBD) and binding sites for HSP90, are thought to have repressive effects on covalently linked, neighboring domains in the absence of their cognate ligands, and binding of the appropriate ligand to an HBD results in de-repression (Picard, 1993). This mechanism was taken advantage of by designing a transcription factor in which a constitutively active transactivating function was regulated by the regulatory region of the rat GR in cis (Picard et al., 1988; Rusconi and Yamamoto, 1987). A chimeric transcription factor comprising the DNA-binding domain of the yeast transcription factor GAL4 (Keegan et al., 1986) and the transactivating domain of the herpes viral protein VP16 (Triezenberg et al., 1988) was chosen as a constitutively active transactivating function. The chimeric protein GAL4-VP16 was thought to act as a strong transcription factor in all cell types because the activation domain of VP16 is known to interact directly with general transcription factors, which are thought to be evolutionarily conserved among eukaryotes (Goodrich et al., 1993; Lin et al., 1991; Sadowski et al., 1988). It has been shown that the regulatory region of the human estrogen receptor could regulate similar chimeric transcription factors in yeast and animal tissue culture cells (Braselmann et al., 1993; Louvion et al., 1993). The regulatory region of the rat GR was added to the chimeric transcription factor and the resulting hybrid transcription factor was designated ‘GVG’ because it consists of one domain each from GAL4, VP16 and GR. A DNA fragment encoding the GVG transcription factor was placed between the cauliflower mosaic virus 35S promoter (Odell et al., 1985) and the poly(A) addition sequence of the pea ribulose bisphosphate carboxylase small subunit gene rbcS-E9 (Coruzzi et al., 1984). As a binding site for GVG, a DNA fragment containing six copies of the GAL4 UAS (Giniger et al., 1985) was fused 5′ to the minimal CaMV 35S promoter (−46 to +9).
Genetic analysis is one of the most important cornerstones upon which the modern life sciences have been built. Historically, genetic studies are largely based on screen for loss-of-function mutations, and this approach is at present still the primary tool for genetic dissection of a pathway. Loss-of-function screens, however, have two major disadvantages. First, this type of screen is incapable of identifying genes that are functionally redundant. Genetic and functional analyses of the ethylene signaling pathway illustrated such an example. Several receptor-like histidine kinases have been identified in Arabidopsis, and they show high homology to each other. These proteins were suggested to be involved in the ethylene signaling, likely to serve as the receptors for the hormone. Whereas none of the null mutations in these genes had any apparent phenotype, transgenic plants carrying 35S-antisense transgenes for all these genes show some loss-of-function phenotype for the ethylene response (Hua and Meyerowitz, 1998). However, dominant-positive or gain-of-function mutations in any of these genes lead to constitutive repression of the ethylene response. As the genomic sequence projects have revealed the presence of many multicopy genes in a variety of species (Lin et al., 1999; Mayer et al., 1999), the problem of functional redundancy has become more apparent. A second limitation for the loss-of-function screens is due to the fact that some mutations cause gametophytic or embryonic lethality, rendering it extremely difficult or even impossible to identify such a gene or a mutation. Many of the Arabidopsis embryo-defective (emb) and related mutants, for example, were identified by microscopic dissection of individual embryos by Meinke and coworkers (Meinke, 1985; Meinke, 1995), indicating technical difficulties for such screens.
As an alternative, screens for dominant-positive or gain-of-function mutations have been developed and used in recent years. In plants, the screen of gain-of-function mutations, also known as activation tagging, was first attempted by Hayashi et al. (1992), who used four copies of the 35S enhancer to activate genes near a T-DNA insertion carrying the enhancer. The most successful example was the identification of the Arabidopsis CKI1 (Cytokinin Independent 1) gene, whose overexpression leads to the regeneration of shoots from explants in the absence of external cytokinins (Kakimoto, 1996). More recently, similar activation tagging constructs have been used to generate a large number of transgenic Arabidopsis plants, from which about 30 dominant mutants have been isolated (Weigel et al., 2000). Analogous to the loss-of-function screens, the main drawback of activation tagging is lethality due to constitutive overexpression of some genes, thus making it incapable of identifying these genes. Indeed, only mutations related to morphological alterations or flowering time were isolated from this large scale screen (Weigel et al., 2000), suggesting that certain dominant mutations, particularly those that severely affect plant development (e.g., embryogenesis), are most likely not recoverable by such methods.
Whereas activation tagging may probe functional significance of some genes, loss-of-function mutations can provide more direct insights on the functions for most genes. Therefore, the combination of both gain- and loss-of-function approaches should be most powerful during the post-genomic era. In this disclosure, we set forth a novel strategy to generate plant mutants that carry both conditional Gain- and Loss-of-Function, termed GLF, mutations in a single genetic locus. The gain- or loss-of-function of a target locus will be reciprocally and tightly controlled by the XVE chemical-inducible expression system, thus enabling phenotype expression of a target locus at a given developmental time of interest. The controllable expression of both gain- and loss-of-function phenotype in a target locus will allow more comprehensive understanding of the gene function compared to the use of individual approaches. In principle, this method is more applicable to species in which high frequency homologous recombination is possible, e.g., mammalian and yeast cells. This can be done by specifically disrupting a natural promoter and replacing it with an inducible promoter that is appropriately functional in mammalian and yeast cells.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the text and respectively grouped in the appended List of References.